Fast and label-free intraoperative discrimination of malignant pancreatic tissue by attenuated total reflection infrared spectroscopy

Abstract. Significance Pancreatic surgery is a highly demanding and routinely applied procedure for the treatment of several pancreatic lesions. The outcome of patients with malignant entities crucially depends on the margin resection status of the tumor. Frozen section analysis for intraoperative evaluation of tissue is still time consuming and laborious. Aim We describe the application of fiber-based attenuated total reflection infrared (ATR IR) spectroscopy for label-free discrimination of normal pancreatic, tumorous, and pancreatitis tissue. A pilot study for the intraoperative application was performed. Approach The method was applied for unprocessed freshly resected tissue samples of 58 patients, and a classification model for differentiating between the distinct tissue classes was established. Results The developed three-class classification model for tissue spectra allows for the delineation of tumors from normal and pancreatitis tissues using a probability score for class assignment. Subsequently, the method was translated into intraoperative application. Fiber optic ATR IR spectra were obtained from freshly resected pancreatic tissue directly in the operating room. Conclusion Our study shows the possibility of applying fiber-based ATR IR spectroscopy in combination with a supervised classification model for rapid pancreatic tissue identification with a high potential for transfer into intraoperative surgical diagnostics.


Supplementary Figure 1 Beam path and penetration depth calculations.
(A) The beam path inside the ATR crystal was calculated and is presented accordingly. Solid red lines indicate the perpendicular beam path from the fiber to the ATR crystal surface. Dashed red lines indicate the beam path due to the numerical aperture of the fiber (the effective numerical aperture of the fiber is 0.3). α half of the top angle of the prism cone, φ angle of incidence to the surface between the crystal and the sample, h height, w width of the crystal. (B) Depth of penetration for different angles of incidence φ. Angle of incidence φ1=27˚ when the beam path from the fiber to the ATR crystal surface is perpendicular, angle of incidence φ2=23˚ and φ3=31˚ when the beam due to numerical aperture of the fiber bends. dp1, dp2 and dp3 depth of penetration respectively for angles of incidence φ1, φ2 and φ3. n1, n2 refractive indexes for germanium (Ge) and blood.
(C) Calculation of depth of penetration for different wavenumbers and angles of incidence. " wavenumber.

Supplementary Figure 2 Illustration of exclusion criteria for spectra.
Calculated absorbances at 1162 cm -1 and 1744 cm -1 identifying spectra associated with high amounts of fatty tissue are indicated. Respective spectra were excluded from further analysis.  Spectral IR absorption bands of pancreatic tissue in the spectral region from 950 cm -1 to 1800 cm -1 and their molecular assignments 16 . δ deformation, ν stretching, s symmetric.

# Sample
Comments in the histological report

N07
Patient received adjuvant chemotherapy (CTx) for a previous gastric cancer several years before and neoadjuvant CTx for the recurrent disease. Cancer cells spread separately into pancreatic tissue -macroscopic "normal" tissue might be invaded by singular gastric cancer cells.

N09
Patient received neoadjuvant CTx, tumor was accompanied by lymphangiosis carcinomatosa, and tumor bulks reached the resection margin -macroscopic "normal" tissue might be coupled with microscopic tumor bulks.

N17
Patient had chronic pancreatitis with inflammatory tissue reaching into the resection marginmacroscopic "normal" tissue might be associated with histologically inflammatory tissue.

N20
Patient had a rare acinar cell carcinoma of the pancreatic tail infiltrating into the spleen, stomach and vascular structures. The underlying pathology might be peri-tumoral pancreatitis.

N26
The analyzed specimen was H&E-stained and re-evaluated with inflammatory tissue within the section typical for pancreatitis.

N27
The analyzed specimen was H&E-stained and re-evaluated with inflammatory tissue within the section typical for pancreatitis.

N31
Patient had an undifferentiated carcinoma with diffuse tumor cell infiltration within the resection margin. Macroscopic "normal" tissue might be coupled with microscopic tumor bulks.

N33
Patient received neoadjuvant chemotherapy before surgical resection. Pathological report states chronic pancreatitis within the tumor-surrounding tissue.

T09
Patient had cholangiocellular carcinoma with microscopic intraepithelial neoplasms reaching into the resection margin -it is macroscopically difficult to differentiate between normal and tumor tissues, and the pathologic report did not mention pancreatitis.

T12
The tissue sample was acquired by punch biopsy during index surgery, and consecutive punch biopsies were also microscopically classified as benign. However, pathologically proven PDAC was resected during subsequent operation.

T29
Patient received neoadjuvant chemotherapy before surgical resection. Most of the tumor cells were necrotic and avital. Peritumoral pancreatic tissue displayed fibrosis.

T40
The analyzed specimen was H&E-stained and re-evaluated with no tumor cells within the section.

Supplementary Table 2 Retrospective review of the misclassified samples.
Pathological reports of misclassified normal (N) and tumor (T) tissue samples of the test set were reviewed for potential explanation of the algorithm error.

Original patient number
Training set - Figure